Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) (ab99697)

Reviews (1)Q&A (3)References (29)

製品の概要

  • 製品名

    Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP)
    IgG Light chain 二次抗体 製品一覧

  • 由来種

    Mouse

  • ターゲット生物種

    Rabbit

  • 特異性

    ab99697 reacts with the light chain of native Rabbit IgG primary antibodies and does not bind to the reduced and denatured Rabbit IgG heavy chain band (50kD).

  • アプリケーション

    適用あり: ELISA, WBmore details

  • 免疫原

    Rabbit Light Chain

  • 標識

    HRP

製品の特性

  • 製品の状態

    Liquid

  • 保存方法

    Shipped at 4°C. Store at +4°C.

  • バッファー

    pH: 7.40
    Constituents: 50% Glycerol, 50% PBS
  • Concentration information loading...

  • 精製度

    Protein A purified

  • ポリ/モノ

    モノクローナル

  • クローン名

    SB62a

  • アイソタイプ

    IgG2b

  • 軽鎖の種類

    lambda

  • 研究分野

アプリケーション

Our Abpromise guarantee covers the use of ab99697 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

アプリケーション Abreviews 特記事項
ELISA 1/4000 - 1/8000.
WB Use at an assay dependent concentration.

Note: This antibody is not recommended for sensitive or quantitative detection of the reduced and denatured Rabbit Light Chains on Western Blots.

画像

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) (ab99697)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) (ab99697)

    IHC image of beta actin staining in human colon formalin fixed paraffin embedded tissue section*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was incubated with ab8227, 3µg/ml overnight at +4°C. An HRP-conjugated secondary (ab99697, 1/8000 dilution) was used for 1hr at room temperature. The section was counterstained with haematoxylin and mounted with DPX.

    The inset negative control image is secondary-only at 1/4000 dilution.

    *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

  • Western blot - Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) (ab99697)
    Western blot - Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) (ab99697)

    Western blot showing that Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697) specifically binds to light-chains


    Left blot: Coomassie stain showing the rabbit heavy (~50kDa) and light-chains (~25 kDa)

    Lane 1: 10 µg rabbit monoclonal was loaded
    Lane 2: 5 µg rabbit monoclonal was loaded
    Lane 3: 2 µg rabbit monoclonal was loaded

    Right blot: WB using Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697) as secondary antibody at 1/10,000

    Lane 1: 1 µg rabbit monoclonal was loaded
    Lane 2: 0.5 µg rabbit monoclonal was loaded
    Lane 3: 0.25 µg rabbit monoclonal was loaded

  • Western blot - Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) (ab99697)
    Western blot - Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) (ab99697)
    All lanes : Anti-beta Actin antibody - Loading Control (ab8227) at 1 µg/ml

    Lanes 1-6 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (ab27252)
    Lysates/proteins at 10 µg per lane.

    Secondary
    Lanes 1 - 2 : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697) at 1/2000 dilution
    Lanes 3 - 4 : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697) at 1/10000 dilution
    Lanes 5 - 6 : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697) at 1/20000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

  • Immunoprecipitation - Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) (ab99697)
    Immunoprecipitation - Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) (ab99697)
    LAP2 alpha was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to LAP2 alpha (ab5162)and 50µl of protein G magnetic beads (lane 1). The antibody was incubated with the Protein G beads for 10min under agitation. No antibody was added to the control (lane 2). Hela whole cell extract diluted in RIPA buffer was added to each sample and incubated for 10min under agitation. Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab5162. Secodary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
    Bands: 83kDa: LAP2 alpha.
  • Immunoprecipitation - Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) (ab99697)
    Immunoprecipitation - Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) (ab99697)
    Catalase was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Catalase (ab1877) and 50µl of protein G magnetic beads (lane 1). The antibody was incubated with the Protein G beads for 10min under agitation. No antibody was added to the control (lane 2). Hela whole cell extract diluted in RIPA buffer was added to each sample and incubated for 10min under agitation. Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab1877. Secodary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697). Bands: 60kDa: Catalase
  • Immunoprecipitation - Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) (ab99697)
    Immunoprecipitation - Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) (ab99697)
    Rad21 was immunoprecipitated using 0.5mg Hela whole cell extract, 5ug of Rabbit polyclonal to Rad21and 50µl of protein G magnetic beads (lane 1). The antibody was incubated with the Protein G beads for 10min under agitation. No antibody was added to the control (lane 2). Hela whole cell extractdiluted in RIPA buffer was added to each sample and incubated for 10min under agitation. Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab42478. Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697) .. Band: 100kDa: Rad21; Non specific - 50kDa: We are unsure as to the identity of this extra band. Although the predicted band size is 75kDa based on Swiss-prot data, a band of 120kDa has been previously observed. Mol Cell Biol. 2002 Dec;22(23):8267-77 PMID: 12417729. Th
  • Western blot - Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) (ab99697)
    Western blot - Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) (ab99697)
    Lane 1: Rabbit IgG loaded at 1ug/well
    ab99697 diluted to 1:2000

参考文献

This product has been referenced in:

  • Karttunen J  et al. Precipitation-based extracellular vesicle isolation from rat plasma co-precipitate vesicle-free microRNAs. J Extracell Vesicles 8:1555410 (2019). Read more (PubMed: 30574280) »
  • Vogg MC  et al. An evolutionarily-conserved Wnt3/ß-catenin/Sp5 feedback loop restricts head organizer activity in Hydra. Nat Commun 10:312 (2019). Read more (PubMed: 30659200) »
See all 29 Publications for this product

ab99697 を使用した論文を発表された方は、こちらまでお知らせください。データシートに掲載させていただきます。

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1-4 of 4 Abreviews or Q&A

Excellent alternative when heavy chain overlies protein of interest in immunoprecipitation

 Excellent
Abreviews
abreview image
Application
Immunoprecipitation
My protein of interest is slightly larger than heavy chain making the use of usual (H+L) secondaries difficult for WB following immunoprecipitation. The two images exemplify the problem. The first is using a secondary that detects both heavy and light chain. Heavy chain band covers my protein of interest. The second image was obtained after stripping the membrane and repeating the WB, this time using ab99697. Now the heavy chain band in the IP lane has disappeared and is faintly existent from the previous experiment in the IgG lane.

Good sensitivity as well. This example is 1:5000, but now I am using 1:10,000.

Excellent!

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投稿 Jul 17 2014

Question

Does this ab recognize the denature form of the primary antibody or not?

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Answer

The has been tested to detect their specific proteins by Western blot under reducing and denatured conditions. Therefore they can recognise their targets in denatured conditions.

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Question

I need to do IP for a protein with expected size of 50KD, I have an primary anti-rabbit for my protein, I don’t have another antibody from different species to do the western blot with different antibody, I have to use same antibody for IP and western blot detection, in the western blot I get a thick band of IgG at 50KD which is masking my band of interest, I was wondering if you could help me to select a secondary which can be used for this experiment and eliminating this thick band in the western blot.

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Answer

This ab appearsto be exactly what I assumed we did not have. The data does suggest that it will solve your problem, if your samples are reduced and denatured.

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Question

I am thinking of buying this antibody to use in western blots after an immunoprecipitation. The antibody used in the immunoprecipitation is a mouse monoclonal. I have tried to use a rabbit polyclonal on the blot to avoid the "heavy IgG band problem" as my antigen is very close in size to the heavy chain. I used ab6721 for this, but it corss-reacts quite heavily with the mouse heavy IgG. I was therefore wonering if you would reccomend the monoclonal light chain specific secondary (ab99697) to avoid this problem. The main concern I have is that with the denatured IgG in the blot being a mouse IgG, I may end up with even worse cross reactivity than before..

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Answer

The rabbit light chain-specific antibody ab99697 should be suitable for your Western blot and I wouldn't expect it to cross-react with the reduced denatured mouse IgG heavy chain, however this antibody has not been tested for reactivity with mouse IgG so we can't guarantee it. However, we do have pre-adsorbed secondary antibodies that have been treated to remove any species cross-reactive antibodies. For an anti-rabbit IgG pre-adsorbed secondary antibody, I would recommend ab7090- https://www.abcam.com/Rabbit-IgG-secondary-antibody-H-L-Pre-Adsorbed-ab7090.html

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